In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted by the sample. The focus of the illumination light beam is moved in an object plane with the aid of a controllable beam-deflection device, generally by tilting two mirrors, the deflection axes usually being mutually perpendicular so that one mirror deflects in the x direction and the other deflects in the y direction. The mirrors are tilted, for example, with the aid of galvanometer control elements. The power of the light coming from the object is measured as a function of the position of the scanning beam. The control elements are usually equipped with sensors to ascertain the current mirror setting.
Especially in confocal scanning microscopy, an object is scanned with the focus of a light beam in three dimensions.
A confocal scanning microscope generally comprises a light source, focusing optics by which the light from the light source is focused onto a pinhole (the so-called excitation aperture), a beam splitter, a beam-deflection device for beam control, microscope optics, a detection aperture and the detectors for registering the detection or fluorescent light. The illumination light is usually input via a beam splitter. The fluorescent or reflected light coming from the object travels back via the beam-deflection device to the beam splitter, and passes through the latter in order to be subsequently focused onto the detection aperture, behind which the detectors are located. Detection light which does not originate directly from the focus region takes a different light path and does not pass through the detection aperture, so that point information is obtained which leads to a three-dimensional image by sequential scanning of the object. A three-dimensional image is usually achieved through layer-by-layer imaging. Instead of guiding illumination light over or through the object using a beam-deflection device, it is also possible to move the object while the illumination light beam is static. Both scanning methods, beam scanning and object scanning, are known and widespread.
The power of the light coming from the object is measured at set time intervals during the scanning process, and hence scanned scan-point by scan-point. The measurement value must be assigned uniquely to the relevant scan position, so that an image can be generated from the measurement data. To that end, it is expedient to measure the state data of the adjustment elements of the beam-deflection device continuously at the same time or, although this is less accurate, to use directly the setpoint control data of the beam-deflection device.
It is also possible in a transmitted-light arrangement, for example, to detect the fluorescent light or the transmission of the excitation light on the condenser side. The detection light beam does not then travel to the detector via the scanning mirrors (non-descan arrangement). For detection of the fluorescent light, the transmitted-light arrangement would need a detection aperture on the condenser side in order to achieve three-dimensional resolution, as in the described descan arrangement. In the case of two-photon excitation, however, a detection aperture on the condenser side can be omitted since the excitation probability depends on the square of the photon density (˜intensity2), which is naturally much higher at the focus than in the neighbouring regions. The vast majority of the fluorescent light to be detected therefore originates with high probability from the focus region, which obviates the need for further differentiation, using an aperture arrangement, between fluorescence photons from the focus region and fluorescence photons from the neighbouring regions.
The resolving power of a confocal scanning microscope is dictated, inter alia, by the intensity distribution and the spatial extent of the focal region of the illumination light beam. An arrangement to increase the resolving power for fluorescence applications is known from PCT/DE/95/00124. This arrangement comprises a light source, which generates an excitation light beam of a first wavelength and an emission light beam of a second wavelength, the excitation light beam being focussed onto a first focal region and the emission light beam being focussed onto a second focal region in a sample, which overlaps partially with the first focal region. The excitation light beam excites optically the sample in the first focal region, while the emission light beam generates stimulated emission in the second focal region. Only the spontaneously emitted light from the part of the first focal region in which no stimulated emission has been generated is then detected, so that an improvement in the resolution is achieved overall. The term STED (Stimulated Emission Depletion) has become attributed to this method.
Since then, STED technology has been developed further to the extent that an increase in the resolution can be achieved both laterally and axially, by providing the focal region of the emission light beam with an intensity distribution which vanishes on the inside. Expressed simply, the focal region is, so to speak, internally hollow. Such an intensity distribution can be achieved, for example, with the aid of a λ/2 plate, which is fitted in a Fourier plane relative to the focal plane of the emission light beam, whose diameter is less than the beam diameter and which is consequently illuminated all round. The focal region of the emission light beam must be made congruent with the focal region of the excitation light beam. Only spontaneously emitted light from the region of vanishing intensity in the focal region of the emission light beam will then still be detected. In theory, resolutions far smaller than 100 nm can be achieved with such arrangements.
It is crucially important that the focal regions of the emission light beam and the excitation light beam be made to overlap suitably.
Even well-corrected high-end optical elements have residual aberrations, which are usually negligible in conventional microscopy but become highly significant in the resolution range considered here. In particular, owing to residual chromatic aberrations, the differing wavelengths of the emission light beam and the excitation light beam lead to serious errors. For example, just the axial chromatic aberration of high-end microscope objectives amounts to about 150 nm, and is therefore above the resolving power theoretically achievable with STED. In the case of a beam-scanning system, lateral aberrations are also added to the axial aberrations, so that the overlap region varies both axially and laterally during the scanning movement.